Method of treating a disorder by suicide gene therapy

ABSTRACT

The present invention relates to a method of prolonging the expression of an exogenous gene in a cell transduced with the exogenous gene. The method comprises co-administration of the exogenous gene with a herpes virus gene, whereby such co-administration prolongs the expression of the exogenous gene in the transduced cell. The method is particularly useful as a means of effecting gene therapy.

The present invention relates to gene therapy.

Gene therapy is a term used to describe the transfer of one or moregenes to a cell. Gene therapy may be used to introduce a gene into acell and provide for subsequent expression of that gene to alter thephenotype of the cell. For example the gene product may protect the cellfrom toxic agents, such as chemotherapeutic agents, increase thesensitivity of a cell to a cytotoxic drug, prolong the effect of anagent, either directly or by overcoming some induced or acquiredresistance, correct a genetic defect within a target cell or to confer anovel function or property on a target cell.

Successful gene therapy depends upon the efficient delivery of asuitable gene to a target cell and expression of the gene at an adequatelevel in sufficient target cells to achieve the desired therapeuticendpoint. The duration of expression required will vary betweendifferent clinical settings, but in most settings continued therapeuticbenefit will depend on targeted, continued, high level, stable andprolonged expression.

Several approaches to gene therapy are under investigation which aim toprovide for targeted gene transfer, controlled expression of the genetransferred and enhanced activity of the transferred gene product.

One approach, as described in European Patent Application 90309430.8 inthe name of The Wellcome Foundation Limited, is to use chimeric viralvectors systems to provide for targeted gene transfer. In this approacha chimera incorporating a tissue specific transcriptional regulatorysequence (TRS) linked to and controlling the expression of aheterologous enzyme is packaged in a synthetic retroviral particle andused for administration to a patient. The expression of the heterologousgene in the patient is therefore targeted to the target tissue. Thisapproach has been used to target expression of cytotoxic agents, forexample thymidine kinase, in cancer cell lines but not in non-cancerouscell lines.

Another approach, as described in International Patent ApplicationPCT/EP98/07380 in the name of Novartis-ErfindungenVerwaltungsaesellschaft MBH, relates to cell-specific expression vectorswhich allow for controlled expression of the gene transferred. Thisapproach uses an expression vector that contains at least one geneessential for replication of the vector under the control of aheterologous transcriptional regulatory system to produce an expressionvector whose replication is controlled by the presence of an agent whichcontrols the activity of the transcriptional regulatory system. Thisallows the gene expression from the expression vector to be modulated incells. If, for example vector replication proceeds at levels that areundesirable the approach allows the level of replication to be reduced.This approach has been used to produce an adenovirus vector containingthe herpes simplex virus type 1 thymidine kinase gene (ad HSV-1 TK)whose replication is modulated in the presence of ganciclovir (GCV). Thevector was administered to subcutaneous tumours induced in a group ofmice and after a period of 5 days GCV given to some of the group ofmice. Immunohistochemistry of all sacrificed mice showed that HSV1 TKexpression was the same between the groups whereas the mice treated withGCV showed diminished vector expression.

A further approach, as described in International Patent Application No.PCT/US98/21672 in the name of Darvin Molecular Corporation relates toexpression vectors transferring genes which encode proteins which aremutated with respect to the wild type protein to have increasedactivity. Such vectors provide a gene product which has enhancedactivity within cells transduced with such vectors.

It has been previously proposed that gene therapy may be useful in thetreatment of neurological diseases such as Parkinson's disease.Alzheimer's disease and brain tumours. Numerous groups are attempting todevelop vector systems that will allow the deliver of potentiallytherapeutic agents to terminally differentiated neurones within theintact brain. It is also an aim to target gene expression to other braincell types (e.g. astrocytes and microglia).

The ability of HSV-1 to establish a lifelong latent infection withinneurones has led to interest in its use as a neuronal gene deliveryvector. However, during HSV-1 latency no viral proteins are produced andtranscription from the viral genome is limited to a family of nuclearRNAs, the latency-associated transcripts, whose function is not wellunderstood. Although HSV-1 vectors which express in dorsal root gangliahave been achieved, whether latency can be achieved in cells of theforebrain is yet to be determined.

One viral vector which is particularly used in current gene therapytechniques under study is the adenovirus vector. The ability of theadenovirus vector to transduce most cell types efficiently has resultedin gene therapy trials involving local administration of adenoviralconstructs, including administration to brain tissues. The complexity ofbrain function and the difficulty in non-invasively monitoring andalterations in gene expression in vivo has meant that the extent andduration of the therapeutic benefit of these trials has been difficultto assess.

Clinical trials of conditional cytotoxic gene therapy of glioblastomaare currently ongoing using retro- and adenoviral vectors encodingHerpes simplex virus-1 thymidine kinase (HSV-1-TK), followed by theadministration of ganciclovir¹⁻⁴. Much of the efficiency of suicide-genetherapy is thought to be due to the ‘bystander effect’, of whichinflammation and anti-tumour immune stimulation appear to be crucialcomponents⁵⁻⁷. In spite of many experimental studies examining theefficiency of suicide-gene therapy-induced glioma regression⁸⁻¹⁷, thereis no information on the incidence of subsequent chronic braininflammation. It has been reported previously that brain gene transferusing adenoviral vectors induces acute, short-lived, inflammatoryreactions¹⁸⁻²⁰, although peripheral readministration of viral vectorsinduces a delayed type hypersensitivity reaction¹⁹, which eliminatestransgene expression, and is accompanied by localised demyelination.Likewise, most transgenic protein expression is mostly, though notexclusively, restricted to the injection site. Such experiments havefailed to demonstrate widespread expression of transgenic proteins inthe brain beyond two months^(18,19,21-24). Understanding the long-termconsequences of suicide gene therapy of brain tumours is thus of crucialimportance.

It is an aim of the present invention to obviate or mitigate adisadvantage of known gene therapy strategies and to provide a methodfor prolonged and widespread expression of a gene of interest. A furtheraim of the present invention is to provide an improved gene therapytreatment.

According to the present invention in a first aspect there is provided amethod of prolonging the expression of an exogenous gene in a celltransduced with the exogenous gene, the method comprisingco-administration of the exogenous gene with a herpes virus gene,whereby such co-administration prolongs the expression of the exogenousgene in the transduced cell.

According to the present invention in a second aspect there is provideda method of prolonging the expression of an exogenous gene in a celltransduced with the exogenous gene, the method comprisingco-administration of the exogenous gene with a conditionally cytotoxicviral gene, whereby such co-administration prolongs the expression ofthe exogenous gene in the transduced cell.

The invention is based on the inventors' study of the long-term outcomesof adenovirus-mediated conditionally cytotoxic gene therapy in asyngeneic glioblastoma model. CNS-1 cells²⁵ were implanted into thestriatum of Lewis rats, and followed by the injection of adenovirusexpressing HSV1-TK, and systemic ganciclovir (GCV). The treatment wasvery efficient, resulting in the survival of 80-100% of animals for atleast 3 months. Unexpectedly, examination of the brains of long-termsurvivors revealed the presence of chronically active braininflammation, as well as very strong and widespread HSV1-TKimmunoreactivity. These data have important implications for the designand evaluation of clinical gene therapy trials of glioblastomamultiforme.

Based on the results of these experiments the inventors went on toinvestigate what gave rise to the prolonged and widespread HSV1-TKexpression and showed that this was not due to the action of the tumourcells, the adenovirus alone or to the action GCV and therefore proposedthat the sustained expression of HSV1 TK may be a previously unknownproperty of HSV-1 TK which may be applicable to all herpes virus genesor alternatively to all viral conditionally cytotoxic genes.

Accordingly, the inventors propose that the expression of any exogenousgene in a transduced cell may be prolonged if the exogenous gene isco-administered with a herpes virus gene or a viral conditionallycytotoxic gene. This proposal should have enormous impact on the fieldof gene therapy where it is important to have prolonged expression of agene of interest. The method according to the present invention thus hasutility in a wide range of conditions from cancer to conditions wheregene therapy is being considered, such as Parkinson's disease and muscledegeneration for example.

An exogenous gene is defined as a gene or gene fragment which isprovided to a transduced cell. The exogenous gene may be one that is notnormally expressed in the cell which is transduced. Alternatively, theexogenous gene may be one that is expressed by the cell that istransduced.

It is well known that genes may be introduced into cells in variousways.

The exogenous gene and/or herpes virus gene may be transferred to thecells of a subject to be treated by transfection, infection,microinjection, cell fusion, protoplast fusion or ballistic bombardment.For example, transfer may be by ballistic transfection with coated goldparticles, liposomes containing the DNA molecule, viral vectors (e.g.adenovirus) and means of providing direct DNA uptake (e.g. endocytosis)by application of plasmid DNA directly to an area topically or byinjection.

According to one embodiment of the first aspect of the present inventionthe exogenous gene and herpes virus gene or conditionally cytotoxic genemay be introduced into the cell as “naked” genes by standard physicalmeans including direct endocytotic uptake.

The “naked” exogenous gene, herpes virus gene/conditionally cytotoxicgene or both may be delivered to the cell as separate pieces of nucleicacid. If the exogenous gene and herpes virus gene/conditionallycytotoxic gene are delivered to the cell together they may be providedon one piece of DNA as a chimera encoding the two full length geneproducts. The “naked” DNA for the exogenous gene and/or herpes virusgene/conditionally cytotoxic gene may be further incorporated within aliposome or virus particle for delivery to a cell.

According to an alternative embodiment of the first and second aspectsof the present invention, the exogenous gene, herpes virusgene/conditionally cytotoxic gene or both may be introduced into thecell via a recombinant vector delivery system. The vector may forexample be a plasmid, cosmid or phage. Such recombinant vectors arehighly useful according to the first and second aspects of the presentinvention for transducing cells with the exogenous gene and the herpesgene/conditionally cytotoxic gene.

The exogenous gene and herpes virus gene/conditionally cytotoxic genemay be co-administered to a cell in one vector or in two separatevectors. If provided in one vector the two genes may be arranged toprovide a linked transcript under the control of the same regulatoryelements if present or may be provided in separate regions of thevector. If provided in two separate vectors it is preferred that thevectors should be arranged to transduce the same cell population.

A vector refers to an assembly which is capable of directing theexpression of a gene. Recombinant vectors may also include functionalelements. For instance, recombinant vectors can be designed such thatthe vector will autonomously replicate in the nucleus of the cell. Inthis case, elements which induce DNA replication may be required in therecombinant vector. Alternatively the recombinant vector may be designedsuch that the vector and exogenous gene and/or herpes virusgene/conditionally cytotoxic gene integrate into the genome of a cell.In this case DNA sequences which favour targeted integration (e.g. byhomologous recombination) are desirable. Recombinant vectors may alsohave DNA coding for genes that may be used as selectable markers in thecloning process.

The recombinant vector may also further comprise a promoter or regulatorto control expression of the gene as required. The recombinant vectormay further be a virion or a transcriptionally targeted vector whichspecifically restricts expression to a particular tissue or cell type.

The exogenous gene and or herpes virus gene may (but not necessarily) beone which becomes incorporated in the DNA of cells of the subject beingtreated. Undifferentiated cells may be stably transduced leading to theproduction of genetically modified daughter cells (in which caseregulation of expression in the subject may be required e.g. withspecific transcription factors or gene activators).

Preferred vector systems for use according to the first aspect of thepresent invention are viral vector systems in which the vector isderived from a DNA virus, for example, parvovirus, picornavirus,pseudorabies virus, hepatitis virus A, B or C, papillomavirus,papovavirus (such as polyoma and SV40) or herpes virus (such asEpstein-Barr Virus, Varicella Zoster Virus, Cytomegalovirus, HerpesZoster and Herpes Simplex Virus types 1 and 2), an RNA virus or aretrovirus, such as the Moloney murine leukemia virus or a lentivirus(i.e. derived from Human Immunodeficiency Virus, Feline ImmunodeficiencyVirus, equine infectious anaemia virus. etc.).

A particularly preferred vector system for use according to the firstaspect of the present invention is the adenovirus vector system. Apreferred adenovirus vector system is described in InternationalApplication No. PCT/EP98/07380.

The viral vector particles comprising either the exogenous gene, theherpes virus gene/conditionally cytotoxic gene or both may beadministered to a host. The host may be an animal host, includingmammalian, non-human primate, rodent and human hosts.

The viral particles may be administered in combination with apharmaceutically acceptable carrier suitable for administration to apatient. The carrier may be a liquid carrier (for example salinesolution) or a solid carrier, such as for example microcarrier beads.

The herpes virus gene which is co-administered to prolong expression ofan exogenous gene in a cell is preferable any gene encoded by the genomeof the family Herpesviridae. Representative examples of this familyinclude Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2,Varicella Zoster Virus, human and murine cytomegalovirus, Pseudorabiesvirus, Marek's disease virus, cercopitecine herpes virus and EpsteinBarr virus.

If the herpes virus gene is to be administered on a herpes viral vectorit is preferred that the herpes virus gene is heterologous to the herpesvirus vector. The term “heterologous” means that the herpes virus geneis not found naturally in the native herpes vector.

A preferred herpes gene that is administered to prolong gene expressionof a co-administered exogenous gene is a herpes virus thymidine kinasegene or variant thereof. The herpes virus TK gene encodes a viral TKprotein which is important in the synthesis of nucleic acid precursorsnormally within cells infected with herpes virus.

In herpes virus infected cells TK can phosphorylate the guanosineanalogue ganciclovir (GCV) resulting in GCV-monophosphate, in contrastto uninfected cells which contain a cellular TK gene which does not acton GCV. GCV monophosphate, if produced is phosphorylated byintracellular protein kinases producing a GCV-triphosphate in cellswhich contain the herpes virus TK gene. The GCV-triphosphate ispreferably incorporated into the DNA of rapidly dividing cells (e.g.cancer cells) but due to its chemical structure cannot promote furtherelongation of nascent DNA resulting in chain termination and cell death.

Various mutant forms of herpes virus TK have been proposed all of whichhave varying degrees of TK activity. The use of such mutants, asdescribed in International Patent Application No. PCT/US98/21672 isenvisaged within the scope of the first aspect of the present invention.

Other herpes virus genes that are preferred to prolong gene expressionof a co-administered exogenous gene is herpes virus ribonucleotidereductase, an enzyme involved in purine metabolism.

It may be that the addition of a pro-drug to the cell transducedaccording to the method of the first or second aspects of the inventionmay enhance the prolonged expression of the exogenous gene. Therefore,preferably, the method according to the first and second aspects of theinvention further comprises addition of a pro-drug to the transducedcell.

Examples of suitable pro-drugs include nucleoside analogues which may bepro-drugs activated by herpes virus TK or other, non herpes virusconditionally cytotoxic enzymes include purine arabinosides andsubstituted pyrimidine compounds, for example as described in publishedEuropean Patent Application EP-A415 731. Representative examples ofnucleoside analogues include GCV, aciclovir, trifluorothymidine,1-[2-deoxy, 2-fluoro, β-D-arabino furanosyl]-5-iodouracil, ara-A, ara-T.1-β-D-arabinofuranoxyl thymine, 5-ethyl-2′deoxyuridine,5-iodo-5′-amino-2,5′-dideoxyuridine, idoxuridine, AZT, AIU (5-iodo-5′amino 2′,5′-dideoxyuridine), dideoxycytidine and Ara-C.

As described above herpes virus TK is a conditionally cytotoxic enzymewhich can act on a non-toxic compound, GCV (a nucleoside analoguepro-drug) to produce a compound which is toxic to a cell, GCVtriphosphate (a nucleoside analogue drug).

Other non-herpes virus genes which encode an enzyme which isconditionally cytotoxic and act on a pro-drug to produce a drug will beknown to persons skilled in the art, and are included within the scopeof the second aspect of the invention. Such conditionally cytotoxicenzymes include thymidine kinase from sources other than herpes virus,carboxypeptidase G2, alkaline phosphatase, penicillin—V amidase andcytosine deaminase gene.

Other nucleoside analogues which may be pro-drugs activated by herpesvirus TK or other, non herpes virus conditionally cytotoxic enzymesinclude purine arabinosides and substituted pyrimidine compounds, forexample as described in published European Patent Application EP-A415731. Representative examples of nucleoside analogues include GCV,acyclovir, trifluorothymidine, 1-[2-deoxy. 2-fluoro. β-D-arabinofuranosyl]-5-iodouracil, ara-A, ara-T, 1-β-D-arabinofuranoxyl thymine,5-ethyl-2′deoxyuridine, 5-iodo-5′-amino-2,5′-dideoxyuridine,idoxuridine, AZT, AIU (5-iodo-5′ amino 2′,5′-dideoxyuridine),dideoxycytidine and Ara-C.

The nature of the exogenous gene to be co-administered with the herpesvirus gene/conditionally cytotoxic gene for prolonged expression willdepend upon why prolonged expression is desired. If for example themethod is for gene therapy of a particular disease the exogenous genewill be a therapeutic gene whose expression is known to be associatedwith treatment of that disease.

If the method is used to study the distribution or expression of aparticular gene in a cell, tissue or organ, the exogenous geneco-administered with the herpes virus gene/conditionally cytotoxic genefor prolonged expression is a marker gene.

Preferred genes to be administered according to the first aspect andsecond aspects of the present invention include those encoding glialcell derived growth factor (GDNF), neurotrophic factor (NGF), neurturin,persefin and other members of the Transforming growth factor βsuperfamily, Nurr-1, gli-1, gli-3, brain derived neurotrophic factor,ciliary derived neurotrophic factor (CNTF), amyloid precursor protein,marker genes like β galactosidase, green fluorescent protein(s) (GFP)amongst others, transducing growth factors β1, β2, β3, inhibitors of NFkappaB, anti-apoptotic genes. e.g. bcl-2, bcl-x1, anti-inflammatory andimmune-modulators such as interleukin 1 receptor agonist (IL-Ira),IL-receptor 2. neuropeptide neurotransmitters, e.g. corticotrophinreleasing hormone, substance P, neurokinins, etc.

As the method according to the first and second aspects of the presentinvention may provide for widespread distribution of the herpesgene/conditionally cytotoxic gene and exogenous gene in brain tissues,the method according to the first and second aspects of the presentinvention are proposed to be particularly useful in gene therapy forbrain diseases. In such therapy it is preferred that the exogenous geneencodes a therapeutic agent associated with such brain diseases. Suchgenes may include those provided above.

Accordingly a third aspect of the invention provides for the use of aherpes virus gene co-administered with a heterologous gene forprophylaxis or treatment of a disease associated with body tissues.

Accordingly a fourth aspect of the invention provides for the use of aconditionally cytotoxic gene co-administered with a heterologous genefor prophylaxis or treatment of a disease associated with body tissues.

Particular brain diseases which the third and fourth aspects of theinvention seek to treat include brain tumours, Alzheimer's disease,Parkinson's disease, Huntington's disease, lateral amyotrophicsclerosis, neurodegenerative and neurometabolic disorders, chronic braininfections (e.g. HIV, measles, etc.), pituitary tumours, spinal corddegeneration (both inherited and traumatic), spinal cord regeneration,autoimmune diseases (e.g. multiple sclerosis, Guillain Barre syndrome,peripheral neuropathies, etc.) and any other diseases of the brain knownto persons skilled in the art.

Treatment of diseases associated with tissues of the body other than thebrain are also envisaged within the scope of the third and fourthaspects of the present invention, such as the liver, muscle, etc.

The inventors propose that as well as prolonging gene expression, theco-administered herpes gene/conditionally cytotoxic gene provides morewidespread distribution of a gene administered by gene therapy than inthe absence of the herpes virus gene. Histological studies show that aHSV1 TK gene is expressed in the axons, dendrites and cell bodies ofneurones in the contralateral side to the side of the brain in which aviral vector was injected. This is in sharp contrast to the observedeffects of steroids on adenoviral vectors where the encoded transgeneexpression was found to be local and the majority of the herpes gene wasfound in astrocytes and not in neurones. In the treatment of neuronaldiseases such as Parkinson's disease for example, it is essential thatthe expression of the exogenous gene, for example in this case adopamine receptor or dopamine agonist is in the neurones and notastroslial cells. Accordingly the third aspect of the invention hasparticular utility in the treatment of brain diseases.

The findings of the inventors have implications in currently advisedtreatment regimes involving gene therapy methods. For example, a trialis underway regarding administering an adenoviral vector containing aherpes virus TK gene together with GCV (so-called suicide gene therapy)in a single treatment cycle for GCV of up to two weeks. The methodsunderlying this trial are described in International Patent ApplicationPCT/US98/21672.

According to current thinking it would be assumed that the TKadministered to a patient in a gene therapy method (e.g. by anadenovirus vector) would be only transiently expressed and therefore itwould only be worthwhile providing a GCV treatment cycle for a shortperiod after administration of the TK. According to the inventors'findings the TK expression would be stable and could last for up to 12months from introduction of the TK gene by gene therapy. Thus, based onthese findings the number of cycles of clinical treatment with GCVshould be increased as compared to currently proposed treatment regimes.

According to the present invention in a fifth aspect there is provided amethod for the treatment of a disorder by suicide gene therapycomprising more than one cycle of administration of a cytotoxicpro-drug.

As it has been shown that the herpes virus TK gene is still expressedafter one year it is apparent that current treatment regimes with TK andGCV of one two week cycle of administration of GCV are curtailedprematurely. Additional cycles of administration of GCV at one month,two months, three months, up to or more than 12 months, from theadministration of the TK gene could still give therapeutic effect as theTK gene will still be expressed in vivo.

The invention will now be described by way of example only, withreference to the following drawings, in which:

FIG. 1 shows survival analysis of Lewis rats implanted with CNS-1 cells,and treated with RAd-128 (TK), RAd-127 (ΔTK), Rad-35 (βgal), or vehicle,and GCV.

FIG. 2 shows brain inflammation in long-term suicide-gene therapysurvivors. Abbreviations used in b, d, f, h are: (c) cortex, (w) whitematter, (s) striatum.

FIG. 3 shows loss of myelinated fibres, HSV1-TK immunoreactivity andmacrophage and lymphocyte infiltration of perivascular cuffs. Thedecrease in myelinated fibres in long term suicide-gene therapysurvivors is not due to autoimmune destruction of oligodendrocytes.Scale bars for a-f is shown in (a) equals 350 μm; scale bar for g-h isshown in (g) equals 35 μm; scale bar for (i) equals 40 μm; scale bar for(j)=50 μm; and for (k, l)=30 μm. Abbreviations used in (i) are: (WM)white matter, (STR) striatum.

FIG. 4 shows persistence of HSV1-TK within neurones in long-termsurvivors of suicide-gene therapy. Small black arrows indicate thepresence of labelling in dendritic spines, while the thicker arrowindicates labelled immuno-reactive axonal boutons. Scale bars: (a)=235μm, (b)=50 μm, and (c)=10 μm.

FIG. 5 shows PCR analysis of brain sections from long term suicide-genetherapy survivors. Immunohistochemical staining for HSV-1 TK in a 3months survivor is shown in a coronal section in (a). The results of thePCR detection of vector genome (IVa2), transgene (TK), or replicationcompetent genome (E1B), in brain sections of Rad128 and Rad127 treatedanimals, and which survived for 90 days is shown in (b). A schematicrepresentation of the regions amplified by PCR is shown in (c). Thesites of amplification for primer pairs a/b, c/d and e/f are indicated.Scale bar (a)=2 mm.

FIG. 6 shows immune-mediated elimination of CNS-1 cells does not lead tochronic persistent infiltration of CD4+ or CD8+ T-cells. Sections werephotographed at a low magnification (a, e, i, c, g, k), and at highermagnification (b, f, j, d, h, l). Scale bars for a, e, i, c, g, k shownin (i,k)=1 mm and for b, f, j, d, h, l shown in, (j, l)=0.5 mm.

FIG. 7 shows that injection of RAd-125 followed by ganciclovir leads tochronic sustained infiltration of CD8+ T-cells. Arrows indicate theboundaries of the white matter showing T-cell infiltration.Abbreviations used in a-d are: (c) cortex, (w) white matter, (s)striatum. Scale bar for a-d, shown in (d)=450 μm.

FIG. 8 shows persistence of HSV1-TK within neurons 1, 5 and 12 monthsfollowing injection of adenovirus TK.

FIG. 9 shows widespread distribution HSV1-TK immunoreactivity throughoutthe anterior frontal and cingulate neocortex at 30 days post-vectorinjection, a: right side of this panel illustrates the hemisphereipsilateral to the injection side, b shows boxed area of cingulatecortex at higher power, d shows boxed area of the piriform cortex athigher power, c and e are boxes enlarged from b and d respectively;notice the strong immunolabeling of pyramidal neurons, their dendritesas well as afferent and efferent axonal processes. Scale bars are shownin each of the panels.

FIG. 10 shows widespread distribution HSV1-TK immunoreactivity at thelevel of the anterior striatum at 30 days post-vector injection. Arrowsindicate the exact location of the original injection site. * indicatesthe enlarged lateral ventricle, ipsilateral to viral vector injection.Boxed areas in panel (a) are shown enlarged in (b) (insular cortex), (d)(anterior commissure) and (e) (corpus callosum). Box in (b), is shownenlarged in (c) to illustrate neuronal morphology. Box in (e), is shownenlarged in (f) to illustrate the detailed axonal morphology of axonscoursing along white matter and entering or exiting the overlyingcerebral cortex. Scale bars are shown in each of the panels.

FIG. 11 shows a time-course of HSV-1 TK expression in rat neocortex at 1month (a,d). 3 months (b,e) and 5 months (c,f). Rats illustrated in a-creceived i.p. GCV and rats d-f received i.p. saline. Boxes to the rightof each lettered panel show higher power views to illustrate neuronalmorphology. Scale bar for the lettered (low power) panels is shown in(a), and the scale bar for all higher power views is shown to the panelto the right of (a).

FIG. 12 shows sections from the neocortex of all 6 rats 1 year followingthe injection of adenovirus encoding HSV-1 TK. Panels shown in (a-c)illustrate the neocortex of rats treated with i.p. GCV; panels (d-e)illustrate the neocortex of rats treated with i.p. saline. Notice thatthere is individual variability in the HSV1-TK immunoreactivitydetected. The highest level of immunoreactivity was detected in theanimal illustrated in (d).

FIG. 13 shows co-injection of RAd HSV-TK with RAd β-Galactosidase.Panels shown in a-f illustrate sections taken from the same animal;panels a-c illustrate the forebrain and substantia nigra immunoreactedwith β-galactosidase antibodies. Panels illustrated in d-f, are allserial sections to those shown in panels a-c, and were immunoreactedwith antibodies against HSV-1 TK.

FIG. 14 shows inflammatory brain responses to the injection of RAd128expressing HSV1-TK. ED1 staining of macrophages/microglial cells at 1month (a,d), 3 months (b,e) and 5 months (c,f) post-vector injection isshown. Brains illustrated in a-c were obtained from animals whichreceived i.p. GCV, while those shown in d-f received i.p.saline. CD8staining is shown at 1 month (g,j), 3 months (h,k) and 5 months (i,l)post-vector injection; panels shown in g-i were obtained from animalswhich received i.p. GCV, and those illustrated in j-l receivedi.p.saline. Notice that inflammatory responses appear somewhat increasedin those animals injected with ganciclovir.

FIG. 15 shows immune response to RAd HSV1-TK in the brains of all 6 ratsinjected 1 year earlier with RAd128. Panels shown in a-f illustrate ED1staining of macrophages/microglial cells: those in a-c received i.p.GCV, and those in d-f are from animals which received i.p.saline. Panelsg-l illustrate CD3 staining: panels g-i are from animals that receivedi.p. GCV, and those shown in j-l are from animals which receivedi.p.saline. Arrow in g show mainly brownish coloured haemosiderindeposition.

EXAMPLES Methods

Cell Culture. The rat glioma cell line CNS-1 was kindly provided byProf. W. Hickey (Dartmouth Medical Center, Department of Pathology,Lebanon, N.H., USA)²⁵. The kidney embryonic cell line 293 was obtainedfrom Microbix Biosystems Inc. (Toronto, Ontario, Canada). Themaintenance of the cell lines was described previously^(37,38).

Adenovirus vectors expressing HSV1-TK, HSV1-ΔTK, or β-galactosidase RAdvectors encoding HSV1-TK (RAd-128) and HSV1-ΔTK (RAd-127) under theshort immediate/early human cytomegalovirus (sMIEhCMV) promoter^(37,38)were generated and characterised as described previously²⁷⁻²⁸. Viruseswere purified using double caesium chloride gradient and titres of1×10¹⁰−1×10¹¹ infectious units (IU)/ml, and particle/pfu ratios of 30were obtained as described in detail elsewhere^(17,20,27,28,37,38). Ascontrol virus, a double caesium chloride gradient purified adenovirus,RAd-35, expressing the E. coli β-galactosidase gene driven by thesMIEhCMV promoter, was used³⁸. We have reported elsewhere that in vitro,CNS-1 cells did express the transgene HSV1-TK following infection withadenoviral vectors, and were sensitive to apoptosis induced followingthe addition of ganciclovir to infected cultures^(27,28).

Lipopolysaccharide endotoxin (LPS) assay and replication competentadenovirus (RCA) assays LPS contamination in each RAd stock was assessedby using the amoebocyte horseshoe crab lysate method (E-Toxate assay,Sigma, Poole, Dorset, UK)³⁹. RAd-127 and RAd-128 showed levels of LPSbelow 5 milli-endotoxin units (mEU)/μl; thus, in the 4 μl injected totalLPS was below 20 mEU. β-galactosidase expressing RAd-35, showed levelsof LPS≦r2 mEU/μl; thus, in the 4 μl injected total LPS was ≦8 mEU. Theamount of LPS needed to produce inflammatory responses in the brain isseveral fold above the upper limit of bioactive LPS activity valueobtained in our bio-assays⁴⁰. Our viral volumes injected wereessentially LPS free. RCA presence was tested by the supernatant rescueassay⁴¹. No RCA was detected in 2.6×10⁸ IU of either of the RAd-127 andRAd-128 vector stocks used in our experiments, showing the absence ofRCA in an amount of vector three times higher than the total amount ofinfectious units that were injected in vivo.

In vivo treatment of gliomas. Male Lewis rats (250-300 g) wereanesthetized with halothane (Zeneca Ltd., Macclesfield, Cheshire, UK)and placed in a stereotaxic frame. A burr hole in the skull was madewith a drill 3 mm to the right and 1 mm anterior to bregma. A 5 μlsyringe fitted with a 26 gauge needle was connected to the manipulatingarm of a stereotaxic frame, and 5 or 10×10³ CNS-1 cells (in 3 μl ofphosphate buffered saline (PBS) were injected over a 3 min period intothe striatum at the following location: bregma +1 mm; lateral +3 mm:ventral −4 mm. The needle was left in place for another 5 min beforeremoving.

Viruses were injected into the tumour site three days after tumourimplantation. Using the same anterior and lateral coordinates, 1 μl ofPBS or 1 μl (2×10⁷ IU/μl) of RAd-127, RAd-128 or RAd-35 were injected ateach of the following ventral coordinates: −5 mm; −4.5 mm: −4 mm; −3.5mm. Starting 12 hours after the injection of the viral vector, 25 mg/kgof ganciclovir (GCV) (Cymevene, Roche Products Ltd., Welwyn Garden City,UK) was injected intra-peritoneally twice daily for 7 days. Animalsinjected with RAd-127, RAd-128, RAd-35 or PBS (n=5 per group) weremonitored daily. Any animal showing any sign of morbidity, wasperfusion-fixed and brains were removed for histological analysis.

In other groups of animals not implanted with CNS-1 tumours, the sameamount of RAd-128 was injected into the brain, and was followed byganciclovir or saline administration for 7 days (n=3 per group). Animalswere perfused either at 1 or 3 months post-adenoviral vector injection.

Histological Analysis, Fixation, Paraffin and Plastic Sections

Rats were anaesthetised, and fixed by cardiac perfusion. First, animalswere perfused with approximately 100 ml of Tyrode solution (0.14 M NaCl,2.7 mM KCl, 1.8 mM CaCl₂, 0.32 mM NaH₂PO₄, 5.6 mM glucose and 11.6 mMNaHCO₃), containing heparin (10 units/ml) (CP Pharmaceuticals Ltd.,Wrexham, UK), and this was followed by 250 ml of 4% paraformaldehyde inPBS, pH 7.4. Brains were removed and placed in 4% paraformaldehyde for24 h. Serial Vibratome sections (70 μm) were maintained at 4 oC in PBS.Sections were stained with hematoxylin and eosin, or Luxol fast blue, orprocessed by immunohistochemistry. Alternatively, some animals wereperfused with 1% glutaraldehyde, 2% paraformaldehyde in 0.1M phosphatebuffer, pH 7.4, and post-fixed for 2-3 days. Some of theparaformaldehyde fixed brains were dissected, and tissue blockscontaining the needle track were embedded on paraffin. Serial sectionswere performed on each block to define precisely the location of theinjection area. Five micron thick paraffin sections were stained withhematoxylin and eosin. Luxol fast blue myelin stain, and Bielschowskisilver impregnation for axons. Immunocytochemistry was performed usingthe avidin-biotin technique, using the following primary antibodies:W3/13 (leucosyalin, mouse monoclonal staining of rat T cells, Serotec):OX8 (mouse monoclonal antibodies recognising CD8+ T-lymphocytes,Serotec); and cyclic nucleotide phosphodiesterase (CNPase; mousemonoclonal SMI 91). Glutaraldehyde fixed tissue was dissected into smalltissue blocks containing the injection site. Material was then furtherfixed/stained in 1% osmic acid in phosphate buffered saline, andembedded into Epon; 0.5 μm thick plastic sections were stained withtoluidine blue.

Immunohistochemistry on Vibratome Sections

Immunohistochemistry was performed on free floating sections, asdescribed before^(36,42). Anti-GFAP (Boehringer Mannheim Ltd., Lewes,East Sussex, UK) and anti-vimentin antibodies (Sigma, Poole, Dorset,UK), specific for astrocyte-specific intermediate filaments, were usedto identify astrocytes, and β-tubulin III (Sigma, Poole, Dorset, UK), todetect neurons and their axons. Anti-ED1 antibodies, to identifymonocytes/macrophages/microglial cells, anti-CD3 antibodies to detecttotal lymphocytes, and anti-CD8 antibodies, which detect CD8 positivelymphocytes and NK cells, were from Serotec Ltd., Kidlington, Oxford.UK. HSV1-TK proteins were detected using an anti-HSV1-TK polyclonalantibody (kindly provided by M. Janicot. Rhone Poulenc Rorer, Paris,France). Sections were washed twice with Tris buffered saline (TBS) (50mM Tris; 0.9% NaCl; 0.5% Triton; pH 7.4), incubated for 15 min with 0.3%H2O2, washed three times with 2 ml of TBS, incubated with 10% horsenormal serum (HNS, Life Technologies Ltd., Inchinnan Business Park,Paisley, UK) in TBS for 45 min, and washed briefly for 10 min with 1%NHS in TBS. Sections were then incubated overnight at room temperaturewith primary antibodies at the following concentration: anti-GFAPdilution 1/200, monoclonal anti-vimentin clone V9 (1/1000), anti-ED-1(1/1000), anti-CD3 (1/500), anti-CD8 (1/500), anti-_-tubulin III(1/2000), and polyclonal anti-HSV1-TK (1/1000). Antibodies were dilutedin 1% NHS in TBS. The following day, sections were washed three timeswith TBS before incubation with a 1/200 dilution of the secondaryantibody (rabbit anti-mouse immunoglobulins biotinylated. Dako Ltd.,High Wycombe, Bucks, UK) for 4 h at room temperature. Sections were thenwashed three times with TBS before incubation with Avidin/Biotin complex(Vectastain ABC Kit, Vector Laboratories, Bretton, Peterborough, UK) for3 h at room temperature. Subsequently, sections were washed three timeswith PBS and two additional times with 0.1 M acetate buffer, pH 6.

Staining was developed by incubating the sections for 5 min at roomtemperature with a solution containing equal volumes of: (i) 0.2 Macetate buffer pH 6 containing 48 g/l ammonium nickel sulphate, 4 g/lglucose, 0.8 g/l ammonium chloride, and, (ii) 1 g/l3,3′-diaminobenzidine and 50 mg/l glucose oxidase in distilled water.The staining reaction was stopped by washing the sections two times in0.1 M acetate buffer pH 6 and two additional times in PBS. Sections wereplaced on gelatin coated slides, dehydrated, coverslipped and mounted.

Statistical Analysis

Survival data were analysed by Kaplan-Meier estimator analysis, andcompared using the generalised Wilcoxon test (Prentice-Peto).

Magnetic Resonance Imagining

Proton magnetic resonance imaging of the rat brain was performed with a4.7 T, 15 cm horizontal bore Biospec (Bruker/Oxford Instruments) system,using a 2.5 cm surface coil. During imaging, animals were maintainedunder general anaesthesia by means of a Halothane (Zeneca Ltd.,Macclesfield, Cheshire, UK)/oxygen gas mixture. In order to detect thepresence of tumours in the brain, axial images, of 2 mm slice thickness,were acquired at approximately 2 mm intervals. The position of the sliceof interest, relative to the plane of coil, was selected by applying a90° “hard” pulse of between 35 and 90 μs duration. In preliminarystudies, T₁, T₂, magnetisation transfer contrast (MTC) anddiffusion-weighted pulse sequences were compared, in order to determinethe conditions required to give optimum contrast between tumour andnormal brain tissue. While the tumours were detectable in T₂-weightedimages, MTC^(43,44) provided greater contrast and was used in allsubsequent experiments. The MTC sequence involved the application of apulse of radiation on 1 s duration, with an offset of 5 kHz and anamplitude of 1.5×10⁻⁵ T. In-plane resolution was 240×480 μm for a fieldof view of 6.2 cm. The superiority of this imaging technique in thedetection of gliomas has been demonstrated previously⁴⁵.

Detection of Adenoviral Genome in Brain Sections Using PCR

Adenoviral sequences were detected in free-floating vibratome-cut brainsections using the polymerase chain reaction (PCR). Briefly, sectionswere digested for 24 hours at 37° C. in 10 mM Tris-HCl (pH8), 10 mMNaCl, 25 mM EDTA, 1% SDS and 4 mg/ml proteinase K. The proteinase K washeat inactivated at 95° C. for 10 minutes after which two rounds ofphenol:chloroform:isoamyl alcohol (25:24:1) extraction were carried out.The genomic DNA was then ethanol precipitated with 3M sodium acetate (pH5.2), washed with 70% ethanol and then re-suspended in sterile watercontaining 20 μg/ml DNase-free RNase.

Ad 5 transcription unit IVa2, Ad 5 E1B, HSV-1 TK and β-actin sequenceswere detected using four different primer sets. Primers A and B (FIG. 5c) are specific to the IVa2 transcription unit of the Ad 5 genome andproduce a PCR product of 686 bp. Primers C and D (FIG. 5 c) are specificto the E1B transcription unit of the Ad 5 genome and produce a PCRproduct of 560 bp³⁵. Primers E and F (FIG. 5 c) are specific to HSV-1 TKand produce a PCR product of 365 bp from TK and ΔTK.

Primers G and H have been modified from a method for detecting chickenβ-actin⁴⁶ and produce a PCR product of 340 bp from exon 4 of ratcytoplasmic β-actin. In a 50 μl PCR reaction, 5-10 μl of genomic DNA wasused in a solution containing 1×PCR buffer (Promega, Southampton, UK),200 μM dTTP, 200 μl dTTP, 200 μM dCTP, 200 μM dGTP, 2 mM MgCl₂, 2 ng/mleach primer and 1U Taq polymerase (Promega, Southampton, UK). PCRconditions were 35 cycles of: 30 seconds denature, 30 seconds anneal,and 1 minute extension followed by a further 10 minutes extension. Theannealing temperatures for primer pairs a/b, c/d, e/f and g/h were 56°C., 57° C., 63° C. and 63° C. respectively. The PCR products wereseparated on a 2% agarose gel and visualised on a UV transilluminatorusing ethidium bromide staining.

Sequences were as follows; A: 5′-AAGCAAGTGTCTTGCTGTCT-3′; (SEQ ID NO. 1)B: 5′-GGATGGAACCATTATACCGC-3′; (SEQ ID NO. 2) C:5′-CAAGAATCGCCTGCTACTGTTGTC-3′; (SEQ ID NO. 3) D:5′-CCTATCCTCCGTATCTATCTCCACC-3′; (SEQ ID NO. 4) E:5′-AAAACCACCACCACGCAACT-3′; (SEQ ID NO. 5) F:5′-GTCATGCTGCCCATAAGGTA-3′; (SEQ ID NO. 6) G:5′-CCAGCCATGTACGTAGCCATCC-3′; (SEQ ID NO. 7) H:5′-GCAGCTCATAGCTCTTCTCCAGG-3′. (SEQ ID NO. 8)Peripheral Priming with CNS-1 Cells

CNS-1 cells were treated with 2 μg/ml mitomycin C overnight to arrestcell division. Twenty-five thousand mitomycin C treated CNS-1 cells(primed rats), or PBS (controls), was injected subcutaneously into theflank of Lewis rats (n=4, per each group). Thirty days later all animalswere rechallenged by implanting 5,000 CNS-1 cells into the striatum,unilaterally. All animals primed peripherally with mitomycin C treatedCNS-1 cells survived, while those primed with PBS died by day 30post-tumour implantation. Surviving animals were perfused 90 days afterintracerebral challenge.

Results

Adenovirus Encoding HSV-1 TK Plus Ganciclovir, Inhibits the Growth ofCNS-1 Gliomas Implanted into the Brains of Syngeneic Lewis Rats

Implantation of 5000 CNS-1 cells unilaterally into the striatum of Lewisrats killed animals within 30 days (FIG. 1). Injection of 8×10⁷infectious units (IU) of a replication-defective recombinant adenovirus(RAd) expressing either the full length HSV1-TK gene (RAd-128), or atruncated, biologically active HSV1-TK gene of reduced intrinsictoxicity²⁶⁻²⁸-HSV1-(TK(RAd-127), into the same site at 3 dayspost-implantation, followed by ganciclovir treatment for seven days,almost completely inhibited CNS-1 glioma growth. Animals were monitoredby weekly magnetic resonance imaging (MRI) brain scans to assesstreatment effectiveness. Tumour growth was only seen in a single animaltreated with RAd-127. No MRI, clinical, or anatomical evidence of tumourgrowth was observed in any other animals. Survival at 3 months posttumour implantation was 100% in animals injected with RAd-128 and83-100% in animals injected with RAd-127 (survival of animals injectedwith RAd-127 shown in FIG. 1 was 83%). The survival rates of animalsinjected with either RAd-128 or RAd-127 were significantly better thanof those animals treated with either RAd-35, an adenovirus vectorexpressing (-galactosidase, or vehicle alone (p=0.0079). No significantdifferences were observed in the survival either between vehicle andRAd-35 treated groups (p=0.1232), or between groups of animals treatedwith either RAd-128 or RAd-127 (p=0.3613). In two identical repeatexperiments, no tumour growth was detected in animals treated witheither RAd128 or RAd127.

Chronic Active Inflammation following the Complete Inhibition of CNS-1Tumour Growth by Gene Therapy: Astrocytosis, Microglia/Macrophage andLymphocyte Infiltration, and Loss of Myelinated Fibres.

General histopatlological analysis Long-term (ninety days) survivors inour experimental syngeneic glioma trials were perfusion-fixed, and theirbrains analysed histopathologically for the distribution of glial,inflammatory, and immune cell markers, as well as for the integrity ofmyelin fibres and oligodendrocytes.

Examination of haematoxylin and eosin stained sections revealed thepresence of inflammatory infiltrates (i.e. diffuse hypercellularitywithin the white matter, striatum and perivascular cuffs), and lateralventricle enlargement (also detected by MRI), ipsilateral to tumour andviral vector injection (FIGS. 2 a, b).

Astrocytosis Immunohistochemical staining for the astrocyte markersvimentin (FIGS. 2 c, d) and glial fibrillary acidic protein (GFAP) (FIG.2 e, f) indicated a widespread activation of astrocytes. GFAP isexpressed by astrocytes, and is upregulated upon activation. Vimentin isundetectable in resting adult rodent astrocytes, but is also upregulatedupon activation. Astrocyte activation was bilateral, but was strongestin the ipsilateral subcortical white matter. Vimentin-positive cellsdisplayed typical astrocytic morphology, with perivascular end-feet. Thedistribution of activated GFAP immunoreactive astrocytes was much widerthan the area occupied by vimentin-immunopositive cells. Astrocyteactivation was seen in all animals.

Microglial/macrophage activation and lymphocyte infiltration ActivatedED1 immunoreactive macrophages/microglia, displaying ongoingphagocytosis (i.e. containing tissue debris), were found mainlyipsilaterally, over a more restricted area than that occupied byactivated astrocytes (illustrated in FIGS. 2 g, h). Within theipsilateral subcortical white matter, the area occupied by ED1+, CD3+,leucosyalin+, or CD8+ cells overlapped with the hypercellularitydetected in the hematoxylin and eosin, GFAP or vimentin stained sections(FIG. 2). In the striatum, activated microglia/macrophages were foundsurrounding the needle track, and infiltrating trans-striatal whitematter tracts. Activated microglia/macrophages were distributedthroughout the dorsal and ventral subcortical white matter, the corpuscallosum, and the ipsilateral anterior commissure. Only very few couldbe detected in the contralateral subcortical white matter (see FIGS. 3e, f). Activated microglial/macrophages were also found withinperivascular cuffs (FIG. 3 g), together with, leucosyalin+, CD3+ andCD8+ lymphocytes (FIG. 3 h). Lymphocytes were also found within theipsilateral subcortical white matter, as well as infiltrating striataltissue (FIGS. 3 i, l).

Loss of Myelinated Fibres

The loss of Luxol fast blue staining strongly suggested a substantialreduction of myelinated fibres in the ipsilateral subcortical whitematter (FIGS. 3 a-b), which spread into its ventral extension. Luxolfast blue staining in the injected striatum was weaker than in thecontralateral side, suggesting actual loss of myelinated fibres alsowithin the striatum. Examination of semithin Epon-embedded sections(FIG. 3 j) stained with osmium and toluidine blue to highlightmyelinated fibres, confirmed the loss of myelinated fibres within thesubcortical white matter. This also indicated the presence of increasedextracellular space (due to fibre loss and edema), and an increase incellularity, composed mostly of astrocytes and oligodendrocytes (FIG. 3j-l). The reduced density of myelinated fibres, the presence ofoligodendrocytes (identified using specific CNPase antibodies;illustrated in FIG. 3 k), together with the absence of primarydemyelinated axons, strongly suggests that the loss of myelin fibres issecondary to tissue degeneration, rather than due to primaryimmune-mediated demyelination. The presence of axonal spheroids (FIG. 3j) further suggests ongoing axonal degeneration.

Long-Term Presence of Immunoreactive HSV1-TK Transgene in the Brains ofRats

We assessed the presence of HSV1-TK immunoreactivity in the brains ofanimals surviving tumour gene therapy for 3 months. Surprisingly, verystrong and widespread immunoreactivity was detected (FIGS. 3 c-d, FIG.4, FIG. 5 a). In the ipsilateral hemisphere, strong immunoreactivity wasencountered in an area overlapping with the distribution of ED1+microglia/macrophages within the subcortical white matter. Further, wedetected strong HSV1-TK immunoreactivity throughout the ipsilateralstriatum, both in neurons and axonal processes, as well as throughoutthe contralateral hemisphere (FIGS. 4, 5 a).

Large numbers of HSV1-TK immunoreactive neurons, axons, and synapticboutons, were distributed throughout significant areas of theipsilateral and contralateral cortex (FIGS. 3 c, d: 4 a-c: 5 a).Immunoreactive neurons were mainly of pyramidal morphology, and wereconcentrated in layers II/III and V (FIGS. 4 a-c). This stronglysuggests that cortico-cortically projecting neurons contain high levelsof HSV-1 TK protein. Importantly, brain areas displaying large numbersof strongly immunoreactive HSV1-TK neurons throughout the contralateralhemisphere (outside of the subcortical white matter), proved to becompletely devoid of any ED1+ activated macrophages, or CD3/CD8 positivelymphocytes (see FIGS. 2 g, 3 e-f). Contralateral striata, onlycontained a large number of HSV1-TK immunoreactive axons (FIG. 3 d, FIG.5 a). These most likely represent axons of cortical neurons projectingto lower levels of the neuraxis. Although strong labelling was found inall animals examined, the distribution of labelled cells varied betweenanimals.

To exclude any non-specific immunoreactivity, the following controlswere performed:

-   -   (i) Sections from animals injected with RAd-127 or RAd-128 and        ganciclovir were immunoreacted with secondary antibodies in the        absence of primary antibodies. No positive staining was        observed, indicating that the secondary antibodies were not        cross-reacting with non-specific tissue components.    -   (ii) Sections from animals treated with RAd35 and ganciclovir,        or vehicle and ganciclovir were immunoreacted with primary        anti-HSV1-TK and secondary antibodies. No immunopositivity was        observed. This excludes the possibility that virus and/or        ganciclovir injection might induce the production of an        endogenous protein detected by anti-HSV1-TK antibodies.

Also, viral stocks were tested prior to injection using the supernatantrescue assay and were shown to be devoid of replication competentadenovirus. To confirm that no very low level contamination could havebeen amplified in the brain during the three months of the experiment, aPCR based method was devised to detect the presence of any replicationcompetent virus in the brain. Three regions of the viral genome wereamplified by PCR from the same brain sections used forimmunohistochemistry (FIGS. 5 b, c). The IVa2 region is present in thegenome of vectors, and in the genome of any replication competent virus.The E1B region is present in the genome of a replication competentvirus, and in 293 cells, but not in the E1 deleted vectors. TK sequenceswill be present only in RAd127 and RAd128, and a β-actin sequence wasused as a control for DNA extraction.

We amplified the TK and the IVa2 region, but not the E1B fragment, fromsections of brains injected with either viral vector 3 months earlier(FIG. 5 b). This demonstrates that vector genomes, but not replicationcompetent viral genomes, were present. To confirm that the E1B fragment,if present, could be amplified from brain tissue, a preparation of anunrelated viral vector contaminated with replication competentadenovirus (as assessed using the supernatant rescue assay) was injectedinto the brain. From sections taken from such brains we amplified boththe IVa2 and the E1B region, as expected (FIG. 5 b).

Persistent Lymphocyte Infiltration is Not Seen following Immune-MediatedElimination of CNS-1 Glioma Cells, but does Occur following theInjection of RAd128 and Ganciclovir Treatment.

To determine whether the chronic lymphocyte infiltration andinflammation was caused by (a) the elimination of CNS-1 cells, (b) theadministration of viral vectors expressing HSV1-TK, or (c) thesubsequent administration of GCV, these variables were testedindependently. Peripheral priming of Lewis rats with 25,000 mitomycin-Ctreated CNS-1 cells, protected rats from a lethal intracerebralchallenge with 5,000 CNS-1 cells. Surviving rats were perfusion-fixed 90days following the intracerebral challenge. No tumour, CD8+, or CD4+,cells could be detected (FIG. 6). Only a modest increase of ED1+macrophages/microglial cells was detected, compared to that followingthe inhibition of tumour growth by gene therapy (compare FIGS. 6 a, bwith FIGS. 2 g, h). Thus, immune-mediated elimination of CNS-1 cellsdoes not lead to a prolonged infiltration of lymphocytes into the brain.Intracerebral injection of 8×10⁷ IU of RAd-128 in the absence of CNS-1cells, followed by administration of ganciclovir or saline for 7 days,and perfusion-fixation 1 or 3 months later, led to a chronic braininflammatory infiltration, with higher numbers of CD8+ lymphocytes inanimals treated with ganciclovir (FIG. 7).

Long Term Transgene Expression using RAd-128 Encoding the Herpes SimplexVirus Type 1 Thymidine Kinase Gene Under the Control of a Short PowerfulImmediate Early CMV Promoter.

Animals were injected with 1×10⁸ infectious units (iu) of RAd-128, andeither injected with ganciclovir or saline twice daily for 7 days.Groups of animals were then perfused 1 month, 3 months, 5 months, or 12months later. Animals were perfusion-fixed and brains were cut in serialsections.

Immunostaining with antibodies against HSV1-TK showed very widespreaddistribution of immunoreactive neurons, which were found throughout thestriatum, cortex, and even distant sites such as the substantia nigra(FIG. 8). It is important to notice that not only were neurons labelledon the ipsilateral side, but also on the contralateral side of thebrain. This wide distribution has never been reported with any othertransgene encoded by either adenoviral vectors, or any other viralvector. TABLE 1 Expression and distribution of transgenes acutely andlong term following the injection of RAd-HSV1-TK or RAd-βgal into thebrains of rodents RAd-HSV1-TK RAd-βgal Striatum +++++ ++ Distantcortical sites +++++ − Distant subcortical sites +++++ − Longevity ofexpression +++++ +

The results shown in the above table indicate that following theinjection of RAd-HSV1-TK expression of transgene is very widespread andlong term. Following injection of RAd-⊖gal, transgene expression is onlyseen in the striatum, and only very little can be detected long term.

Discussion

Three main findings were made in the course of our conditional cytotoxicgene therapy studies in a syngeneic rat glioblastoma model: (i) completetumour growth inhibition in the majority of animals; (ii) a chronic,ongoing, inflammatory process, characterised by T-cell andmacrophage/microglial infiltration and activation, and loss ofmyelinated fibres and axons in long-term survivors; and, (iii)transgenic HSV1-TK was still being expressed at very high levels inneurons throughout the brains of survivors ninety days post vectoradministration.

This is the first report of: (i) chronic active inflammation in responseto a single, successful, brain glioma gene therapy regime; and, (ii) thelong term presence of the therapeutic enzyme, HSV1-TK. Furthermore, wedemonstrate that the chronic inflammatory process does not impairlong-term transgene expression in the brain. As the presence of HSV1-TKthroughout the contralateral cortex and striatum did not result in overtlocal inflammatory responses, additional mechanisms will have to beinvoked to explain the usual short lived transgene expression followingadenovirus vector-mediated gene transfer to the brain^(18-21, 23-24).

Long-Term Striatal and Peri-Striatal Inflammatory Responses to theTreatment of Syngeneic Gliomas

Most previous experimental models of glioblastoma gene therapy have usedC6, 9L, or F98 glioma cells8-17, and failed to report any chronicinflammatory responses. Following a single administration of adenovirusencoding the marker gene β-galactosidase or HSV1-TK to either rats,mice, non-human primates, or human glioma, mainly acute, short-lived,and dose-dependent, inflammatory responses have so far beendescribed^(18-21, 23,24, 29-31). The ongoing nature of the inflammatoryprocess detected in our model is supported by our finding ofperivascular cuffs, composed of both T-cells and activatedmicroglia/macrophages. Importantly, injection volumes, viral andganciclovir doses, and our experimental paradigm, are within the rangedescribed in the literature⁸⁻¹⁷. However, this is the first report of asyngeneic glioma gene therapy model in Lewis rats, which are highlysusceptible to experimental allergic encephalomyelitis³².

Previously, localised demyelination has only been described followingthe peripheral readministration of adenovirus vectors¹⁹. Moreover, inour experiments, the chronic inflammatory response was also limited tothe hemisphere originally injected with tumour cells and viruses.Importantly, no inflammatory responses were detected at any distantsites expressing high levels of immunoreactive HSV1-TK. Further, ourdata uncovered a loss of myelinated fibres, edema, and indices ofongoing axonal degeneration, while oligodendrocytes were preserved andprimary demyelinated axons absent. This demonstrates that the loss ofmyelinated fibres, is not primary, or immune-mediated, but secondary totissue injury and axonal loss.

Adenovirus injection into brain parenchyma stimulates the secretion ofIL-1 and IL-6, while injection into the lateral ventricle inducessecretion of IL-1, IL-6, and TNF-α²⁰. Thus, the immune-suppressivemicroenvironment of the brain and gliomas (which express TGF-β³³ andFas-Ligand³⁴) could be modified by viral-mediated gene therapy, throughthe secretion of pro-inflammatory cytokines, coupled to inflammationelicited through HSV1-TK and ganciclovir mediated cell killing. Thiscould enhance tumour immunogenicity and improve gene therapy'santi-tumour activity.

The persistent inflammation is not exclusively due to the elimination ofCNS-1 cells, since the subcutaneous priming with growth arrested CNS-1cells completely protected animals from an intracerebral challenge,without leading to a chronic inflammatory response. Thus, immunesystem-mediated elimination of glioma cells (as opposed to adenovirusmediated gene therapy) does not lead to chronic inflammation andlymphocyte infiltration. Importantly however, injection of RAd-128followed by ganciclovir administration did cause an important influx ofCD8+ cells, which was much reduced in the absence of ganciclovirtreatment. Hence, the persistent inflammation is a result of thecombined effect of HSV1-TK and ganciclovir, but is not a direct resultof the elimination of the tumour cells per se. Whether this effect willbe shown to be specific to adenovirally-encoded HSV1-TK, or whether itwill be seen when HSV1-TK is expressed by other viral vectors remains tobe determined.

Long-Term Presence of the Adenovirally Encoded HSV1-TK Transgene

Another significant finding was the widespread presence ofimmunoreactive HSV1-TK within ipsilateral and contralateral neocortexand striatum, as well as within ipsilateral glia and inflammatory cells3 months following vector injection and ganciclovir administration. So,HSV1-TK immunoreactive cells either became infected following theadministration of ganciclovir, or are resistant to HSV1-TK plusganciclovir dependent cytotoxicity. HSV-1 immunoreactive neuronsdisplayed normal morphologies, suggesting that the long-term presence ofHSV1-TK and ganciclovir administration did not compromise neuronalsurvival. This contrasts with sympathetic neurons in culture, in whichinfection with adenovirus vectors led to neuronal death in a few days³⁵.Our findings also contrast with previously published experimentsdescribing much more anatomically restricted adenoviral encoded neuronalprotein expression^(18,19, 21-24).

The presence of HSV1-TK throughout the ipsilateral and contralateralneocortex was restricted to pyramidal neurons, mainly located in layersII-III and V, which contain callosally and cortico-cortically projectingpyamidal cells. Such neurons may have taken up vectors through axonalvaricosities present on axons coursing throughout the subcortical whitematter overlying the injected striatum³⁶. Altematively, HSV1-TK proteincould have been released by dying cells (if spared from intracellulardegradation) and taken up by axonal terminals to be transportedretrogradely to parent neuronal cell bodies. This is unlikely, however,given the widespread distribution of HSV1-TK protein and viral vectorgenomes, in distant cells, including neurons, throughout the brain. Thepresence of vector genomes, but the absence of replication competentvirus from brains of long term survivors, indicates the apparentstability of, and long term expression from, adenoviral vectors injectedinto the rodent brain.

Implications for Clinical Gene Therapy Trials of Glioblastoma Multiforme

In spite of aggressive surgery, chemo- and radiotherapy, median survivalof glioblastoma patients is below 12-15 months, and has not improvedduring the last 30 years. This calls for novel treatments, such as genetherapy. All current treatments have significant side effects. Surgerycan damage vital brain areas, chemotherapy has very high toxicity, andwidespread demyelination is a long-term consequence of radiotherapy.

The above experiments appear to show the operation of two phenomena.One, is the enhanced distribution of transgene, which is not seen withany other transgene. The second one, is the very long term anddistributed expression of the transgene throughout large areas of thebrain. The basis underlying this phenomenon could be an intrinsiccharacteristic of the HSV1-TK gene, or the mRNA encoding HSV1-TK, or theprotein itself. This intrinsic characteristic may be one found in allherpes virus genes. Further, it could be a characteristic of the genethat is dependent on the viral vector environment. It is possible thatthe widespread distribution and longevity of expression will beconferred onto any second gene co-expressed with a herpes virus gene,for example HSV1-TK, either in the same vector, or an associated vectorthat can infect the exact same cells simultaneously. These hypothesisare now being tested. It is further possible that the administration ofganciclovir confers an advantage, but how big this is, also remains tobe determined.

Several clinical trials of glioblastoma suicide gene therapy usingretro- and adenoviruses encoding HSV1-TK, in combination withganciclovir, are currently ongoing¹⁻⁴. Our work has several importantimplications for clinical trials of glioblastoma gene therapy usingadenoviruses expressing HSV1-TK: (i) effective gene transfer may occurto so far unexpected widespread areas of the human brain; (ii) thedevelopment of a chronic inflammatory response in humans could lead to aloss of myelinated fibres; (iii) long term persistence of HSV1-TK couldlead to improvements in the clinical trial's schedule of gancicloviradministration. Extending the administration of ganciclovir couldimprove the anti-tumour effect by allowing killing of transduced gliomacells that have not yet entered the cell cycle during short periods ofpost-surgical ganciclovir administration in current use. The severity ofgene therapy's untoward effects will have to be balanced with itsincreased anti-glioblastoma efficiency, vis-a-vis the limitations ofcurrently used therapies.

FURTHER EXAMPLES

Further studies to evaluate the spread, level, and longevity of RAdmediated HSV-1-derived-TK expression, driven by the MIE-hCMV promoter inthe Lewis rat brain were carried out and described below. In particularthe aim was to determine whether the high level and widespreadexpression of HSV-1-TK following the administration of an adenoviralvector into the brain seen in the earlier experiments described abovewas due to: (i) the ganciclovir treatment, (ii) tumour presence, or(iii) whether it was transgene dependent. These factors could not bedissected in the original experimental design.

In summary the results of these additional experiments demonstrate that,using the HSV-1 derived TK as a transgene:

-   -   1) Despite an inflammatory immune response, intra-striatal RAd        mediated delivery leads to widespread, high-level and long lived        neuronal transgene expression that is transgene dependent.    -   2) This effect does not depend on ganciclovir treatment of        animals injected with the vector.    -   3) This effect is not dependent upon the co-implantation of        potentially immune-suppressive glioma cells in the brain.    -   4) The effects detected are restricted to the RAd expressing        HSV1-TK, since neither the spread, high level expression, nor        widespread distribution are conferred upon a co-injected vector        expressing a different transgene.

Harnessing this transgene dependent property could increase the spreadand expression levels of therapeutic transgenes, thus improving theirefficacy in the treatment of neurological disorders.

MATERIALS AND METHODS

Vectors:

Experiment 1: Adenoviral vectors used were E1A deleted recombinantadenoviral vectors encoding the full length Herpes Simplex Virus-derivedThymidine Kinase gene⁴⁷ driven by the MIE-hCMV promoter, as describedabove.

Experiment 2: As experiment 1, in addition to a similar vector encodinglacZ (RAd 35) and a RAd encoding HPRT (RAd HPRT). Vectors were titratedby end point dilution, characterised by Southern blot or PCR and werenegative for replication competent virus, as analysed by supernatantrescue assay, and lipopolysaccharide.

Experiment 1:

18 adult male Lewis rats (weight 250-300 g) had 5×10⁷ infectious units(iu) of RAd 128 injected stereotactically into the mid striatum(co-ordinates: anterior to bregma +1 mm; lateral +3 mm; ventral −4 mm)whilst under halothane anaesthesia, using a 25-gauge needle on a 10 μlHamilton syringe. 12 hours later, 9 rats received intra-peritonealinjections of ganciclovir 25 mg/kg twice daily and 9 received i.p.saline injections for 7 days. Four rats, 2 treated with GCV, and 2injected with saline, were sacrificed by perfusion-fixation withTyrode's solution containing 10 units/ml heparin (approx. 200 ml)followed by 250 ml of 4% paraformaldehyde in phosphate buffered saline(PBS) at 1 month, 3 months and 5 months. The remaining 6 rats (3 treatedwith ganciclovir, and three injected with saline) were sacrificed at 1year.

Experiment 2:

To assess the effects of HSV-1 TK on the expression another transgene,lacZ, a combination of either RAd TK+RAd HPRT or RAd 35+RAd HPRT or RAd35+RAd TK was injected into the mid striatum of 9 rats (3 rats pergroup). The total vector dose was 8×10⁷ iu (i.e. 4×10⁷ iu per vector).

Tissue Processing

Rat brains were post fixed overnight in 4% paraformaldeheyde in PBS,then sectioned, using a Leica VT1000S vibrating blade microtome at 50μm. Sections were stained by free-floating immunohistochemistry forHSV1-TK (rabbit polyclonal anti-HSV1-TK, courtesy of M. Janicot, RhonePoulenc Rorer, Paris, France. Dilution 1:400), β-galactosidase (mousemonoclonal, Promega, dilution 1:1000), the macrophage marker ED1 (mousemonoclonal, Serotec, 1:1000 dilution) and cytotoxic T cell and NK cellmarker CD8 (mouse monoclonal, Serotec, 1:500 dilution). Secondaryantibodies used were biotinylated rabbit anti-mouse immunoglobulin orswine anti-rabbit immunoglobulin (Dako, Carpinteria, California,dilution 1:200).

Results

Spread of HSV1-TK

FIGS. 9 and 10 show coronal brain sections at differentanterior-posterior levels along the neuraxis, 1 month following a singleintra-striatal injection of 5×10⁷ i.u. of RAd HSV-1 TK. We detected adiffuse, high level HSV1-TK protein immunoreactivity throughout thestriatum and many areas of the neocortex, both ipsilateral andcontralateral to the injection site. In addition there wasimmunoreactivity in the anterior commissure, nucleus accumbens,ipsilateral nucleus of the horizontal limb of the diagonal band,magnocellular preoptic nucleus and several thalamic nuclei e.g. theparacentral, anteroventral and anteromedial thalamic nuclei (not shown),among others. FIG. 9 illustrates a difference in the distribution ofHSV-1 TK immunoreactivity between the two hemispheres, and anteriorlevels of the neuraxis. Notice the lack of immunoreactivity in theolfactory cortical areas contralateral to the injection side. As opposedto the cingulate, frontal and parietal cortices, this area does not haveaxonal connections with the contralateral cortex or striatum, so vectorcould not reach this area through axonal pathways. Neurons, as well astheir dendritic processes, axons, and axonal terminals could be clearlydetected throughout both hemispheres. The decussating fibres ofcortico-cortical axons, coursing through the corpus callosum could beclearly identified, and were seen branching off and entering theneocortex [FIG. 10].

Longevity of HSV1-TK Immunoreactivity

Cortical HSV1-TK immunoreactivity was maximal at 1 month post-vectoradministration and subsequently declined. FIGS. 11 and 12 show thebrains displaying the strongest levels of immunoreactivity for each timepoint. Importantly, however, staining persisted even at 1 yearpost-vector injection in all 6 brains. HSV1-TK immunoreactivity in thebrains of all animals sacrificed at 1 year are illustrated in FIG. 12.In one brain (FIG. 12 d), cortical staining at 1 year was almost as highas at 1 month (FIG. 11). Striatal staining showed a similar maximum at 1month and a gradual decline with persistence at 1 year (results notillustrated).

Although at 3 and 5 months there appeared to be more neocortical HSV1-TKimmunoreactivity in the GCV treated group, this was not the case ateither 1 month or 1 year. Indeed, at 1 year the highest level ofimmunoreactivity was detected in the brain of an animal injected withsaline. Thus, we conclude that GCV had no effect on the maintenance ofHSV1-TK immunoreactivity in the rodent brain. Furthermore, we did notdetect any major differences in the degree of spread between the GCV andsaline treated groups at any time point.

Co-Injection of RAd HSV-1 TK with RAd 35

To investigate whether the above results were due to a property of theHSV1-TK protein potentiating transgene expression, RAd HSV1-TK wasco-injected with RAd 35, a first generation adenoviral vector expressingthe transgene lacZ, under the control of the exact same. MIEhCMVpromoter [FIG. 13]. The striatal HSV1-TK immunoreactivity indicatesthat, from a technical point of view, the injection in these animals wassuccessful in accuracy and delivery. Retrograde transport and expressionis illustrated by the numerous HSV1-TK immunopositive neurons in thesubstantia nigra pars compacta, known to project to the corpus striatum.However, serial vibratome sections taken from the same site andimmunoreacted for β-galactosidase, showed very little expression,limited to the area immediately around the needle tract of the corpusstriatum with negligible retrograde expression in the substantia nigrapars compacta. A control with RAd 35 co-injected with RAd HPRT showedsimilar β-galactosidase expression.

Inflammatory Response

There was a strong initial immune response to the vector as illustratedby the ED1 (a macrophage and activated microglial marker) [FIGS. 14 a-f]and CD8 (NK cell and cytotoxic T cell marker) [FIGS. 14 g-h]immunoreactivity. Apart from the 1-year time point [illustrated in FIG.15], all brains showed similar levels of inflammation, which graduallydeclined over the year. Despite widespread transgene expression, the ED1and CD8 response was limited to the area of brain tissue surrounding theinjection site. At 1 year, 5 of the brains showed similar levels ofinflammation. Interestingly, one brain showed widespread ED1 staining[FIG. 15 c] associated with enlargement of the ventricles and reducedstriatal volume bilaterally. However, this was not accompanied bycomparable CD8 persistence. Levels of inflammation detected at 1 and 3months post-injection were similar to those reported previously.

DISCUSSION

This paper illustrates the remarkable phenomenon of widespread, highlevel and long term HSV-1 TK expression in the brain when delivered by afirst generation recombinant adenoviral vector. This phenomenon is notdependent upon the promoter or vector, but unexpectedly, the HSV1-TKtransgene itself. It also illustrates the shortcomings of relying ongene expression as an indicator of the efficiency of adenovirustransduction of the brain: RAd-mediated expression of intracellularproteins would be judged much less efficient on the basis of mostresults described in the current literature.

This phenomenon can be explained by a combination of factors. Firstly,since RAds are known to undergo retrograde axonal transport, the patternof staining is likely to due to this, as opposed to diffusion. A recentpaper by Gerdes et al⁴⁸ showed that using the MIEmCMV promoter, muchhigher transgene expression levels could be achieved in the brain,following the injection of very low doses of viral vectors. However,although spread of transgene expression increased, it was still limitedto the ipsilateral striatum. Since the MIEmCMV is a glial specificpromoter, this was not totally unexpected. Even if virus spread to otherparts of the brain by axonal transport, this could possibly not bedetected using MIEmCMV driven transgene expression as a marker for viralspread. These data show, however, that adenovirus can diffuse in thebrain over much larger distances than previously thought.

The most likely explanation that retrograde transport lead to the spreadof RAd-encoded HSV1-TK throughout the brain is illustrated by theimmunopositive axons seen in the corpus callosum, the main ‘decussatinghighway’ of the brain. Furthermore, the piriform cortex contralateral tothe injection site was devoid of HSV1-TK immunoreactivity, while theipsilateral piriform cortex did display strong HSV1-TK staining.Importantly, the piriform cortex is not connected by strongcontralateral cortico-cortical or cortico-striatal connections.Retrograde transport of adenovirus has been described previously, butthe extent detected in our experiments, was unexpected. Thus, there iseither more expression and/or less clearance of the HSV1-TK protein orHSV1-mRNA. Apart from the transgene, the vector and expression cassetteare the same as in our vector RAd35, which expresses β-gal. Thedifference between β-gal and HSV1-TK expression when co-injectedexcludes the possibility of altered anti-vector immune responses leadingto such high levels and widespread HSV1-TK expression. However, it doesnot exclude the possibility that the HSV1-TK protein could be lessimmunogenic. However, this in unlikely since when tested side by sideHSV1-TK was shown to be more inflammatory than β-gal.

The most likely explanation for the high levels of expression is thatthe HSV1-TK transgene is potentiating or stabilising its own expressionlevels above that dependent on the MIE-hCMV promoter. This is supportedby two studies, which showed that the HSV-1 TK gene contains severalshort nucleotide sequences or sub-elements within its translated regionthat facilitate pre-mRNA transport from the nucleus to the cytoplasm.These sub-elements may also act by enhancing transcriptional activation,stabilising mRNA and enhancing translation. The mechanisms by which theyfunction have yet to be fully elucidated. However, at least one of these‘RNA processing enhancers’, an 119 nucleotide sequence isolated by Liuand Mertz, binds in a sequence specific manner to heterogeneous nuclearribonucleoprotein (hnRNP) L, hnRNPs are a group of ribonucleoproteinswhich are known to be involved in the regulation of mRNA transport,turnover and translation.

The widespread transgene expression with HSV1-TK illustrates howimportant either vector and/or transcriptional targeting will be whenusing potentially cytotoxic transgenes in the CNS. On the other hand,harnessing this phenomenon will allow more widespread transgeneexpression in the treatment of global brain disorders.

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1-38. (canceled)
 39. A method for the treatment of a disorder by suicidegene therapy comprising: providing a cytotoxic pro-drug; andadministering more than one cycle of said cytotoxic pro-drug.
 40. Themethod according to claim 39, said administering more than one cycle ofsaid cytotoxic pro-drug is repeated substantially every one month, twomonths, three months, four months, six months, eight months, ten monthsor twelve months or fractions thereof.
 41. The method of claim 39,wherein said pro-drug is selected from the group consisting of GCV,aciclovir, trifluorothymidine, 1-[2-deoxy, β-fluoro, β-D-arabinofuranosyl]-5-iodouracil, ara-A, ara-T, 1-β-D-arabinofaranoxyl thymine,5-ethyl-2′deoxyuridine, 5-iodo-5′-amino-2,5′-dideoxyuridine,idoxuridine, AZT, ATU (5-iodo-5′ amino 2′,5′-dideoxyuridine),dideoxycytidine and Ara-C.